Biologically active lipoprotein and its use

ABSTRACT

A lipoprotein possessing pulmonary surfactant activity comprising an alveolar polypeptide or protein and, covalently bound thereto, one or two fatty acid residue(s); 
     a pharmaceutical composition comprising such lipoprotein and a phospholipid type of material; and 
     a method of facilitating respiration in mammals including man, comprising administering an effective amount of such a lipoprotein or pharmaceutical composition to the respiratory tract of a patient subject to respiratory disorder so as to reduce surface tension at the air-liquid interface of the patient&#39;s alveoli.

This application is a divisional of application Ser. No. 07/423,346,filed Oct. 18, 1989 and now U.S. Pat. No. 5,223,481.

The present invention relates to lipoproteins possessing pulmonarysurfactant activity, i.e. useful as components of pulmonary surfactantcompositions for providing normal respiration in mammals including man.The invention also covers a method of facilitating respiration inmammals including man.

Pulmonary surfactant, which is a phospholipid-protein complex, isessential for normal respiration by reducing surface tension at theair-liquid interface of the alveoli (1). Different surfactant-specificproteins have been detected. One group comprises comparatively largeglycoproteins with molecular weights varying between 28 and 36 kDa,depending on the degree of glycosylation (2,3). This protein is solublein water and the primary structure of the forms from canine and humanlung has been determined (4-6). In the presence of calcium ions thisprotein apparently participates in the formation of surface-activetubular myelin from secreted lamellar bodies (7) and increases the rateof adsorption of surfactant phospholipids (8). Although this proteinprobably is functional for the endogenous surfactant, synthesized in thealveolar epithelial type II cells, it does not seem to be essential forthe physiological activity of exogenous surfactant preparations designedfor replacement therapy (9-11).

A second group of surfactant-specific proteins constitutes forms withlow-molecular weights (≦14 kDa) (2,9,12-20). These proteins are veryhydrophobic and are composed of different proteins which may be soluble(9) or insoluble (2,16,17) in ether/ethanol.

Both proteins require organic solvents for solubilization andpurification, and are heterogenous by multiple start positions in theN-terminal regions (truncated forms). Recombination of either of theseproteins with synthetic phospholipids yields a surfactant preparationwith physical and biological properties which in many respects aresimilar to those of natural pulmonary surfactant.

The two low-molecular weight proteins have unrelated structures andsizes; the smaller form is not a fragment of the larger. Recently, cDNAsegments of the longer form (21) have been described from dog. Thepresent invention concerns lipophilic low-molecular weight apoproteinsof mammalian origin.

Based on extensive scientific research and experimentation andcontradictory to previous scientific theories it has now unexpectedlybeen found, that pulmonary surfactant activity is related to alipoprotein, wherein an alveloar polypeptide or protein has covalentlyattached thereto one or two fatty acid residues. The fact that it hasturned out that lipoprotein is the active component in compositionspossessing pulmonary surfactant activity is an unexpected new discovery,and the scientific theories hitherto launched have all been directed tothe belief that the alveolar proteins of relevance are present inadmixture with phospholipid type of materials, whereas up to now no onehas expected the protein to be covalently associated with anyhydrophobic substances, such as fatty acids.

Based on this surprising finding the present invention thus provides anew and novel lipoprotein comprising an alveolar polypeptide or proteinand, covalently associated thereto, one or two fatty acid residues.

The fatty acids involved are selected from traditional fatty acids from14 to 22, such as those having 16 or 18 carbon atoms, and may beselected from palmitic, stearic, oleic, linoleic and linolenic acids.Palmitic and stearic acid residues are preferred, in particular palmiticacid residues. The lipoprotein of this invention preferably contains twopalmitic acid residues per polypeptide molecule.

The polypeptide constituting part of the new lipoprotain preferablycomprises the minimal amino acid sequence: Ile-Pro-Cys-Cys-Pro-Val.

This amino acid sequence constitutes the consensus region for all knownpolypeptides originating from different mammal species.

A preferred polypeptide comprises the amino acid sequence: ##STR1##

In this sequence X is selected from Gly and Arg, Gly being a preferredamino acid residue.

It is preferred that the polypeptide part of the lipoprotein accordingto the invention is of human, porcine or bovine origin. In regard to thecharacterization of such polypeptides reference is made to Febs Lett.(1988), Vol. 232, No. 1, 61-64. The full disclosure of this report isincorporated herein by reference.

More specifically, the polypeptide comprises the following amino acidsequence: ##STR2##

In this sequence X is again Gly or Arg, preferably the former.

The lipoprotein of the present invention involves polypeptides asoutlined above in their N-terminally truncated forms. Such truncation ispreferably comprised by one or two amino acid residues.

In regard to the positions of attachment of the fatty acid residues ofthe polypeptides defined above they are covalently attached to one orboth of the cysteine residues in positions 5 and 6 of the molecule, suchattachment being such as to form thioesters.

The present invention also involves pharmaceutical compositionscomprising in combination a protein or protein composition as definedabove and a phospholipid type of material. In such pharmaceuticalcomposition the protein is a minor component, and a preferred weightrange of the contents of the composition of the protein is about 0.5 toabout 10% by weight thereof. It is particularly preferred that theprotein constitutes about 1 to 5% by weight of the composition as awhole. As an example of phospholipid material there may be mentionedphospholipids based on palmitic acid. In addition to such phospholipidmatrix the composition of the invention may also contain otheradditives, such as pharmaceutically acceptable carriers or diluents,stabilising agents, and other conventionally used pharmaceuticallyacceptable additives.

The lipoproteins according to the present invention have been found tocontribute significantly to pulmonary surfactant activity. Accordingly,the lipoproteins and compositions of the invention are particularlyuseful as components of pulmonary surfactants. Furthermore, theinvention includes a method for facilitating respiration in mammalsincluding man, such method comprising administering an effective amountof a lipoprotein or composition according to the invention to therespiratory tract of a patient in need of treatment for respiratorydisorders. By such administration it is possible to significantly reducesurface tension at the air-liquid interface of the patient's alveoli.The administration can take place directly into trachea or bronchi, butcan also take place through the oral cavity by using an aerosol spray ofa conventional type.

BRIEF DESCRIPTION OF THE FIGURES

In the following the present invention will be further illustrated bynon-limiting examples. This exemplification will be made with referenceto the appended drawings, wherein

FIG. 1 shows the mass spectrogram of human surfactant lipoprotein;

FIG. 2 shows the corresponding mass spectrogram after treatment withtrimehyl amine;

FIG. 3 shows the corresponding mass spectogram after treatment withtrimethyl amine followed by iodoacetate;

FIG. 4 shows the mass spectrogram after treatment with potassiumhydroxide;

FIGS. 5 and 6 show diagrams on surface activity of artificial surfactantpreparations using the pulsating bubble technique.

The lipoproteins according to this invention are highly hydrophobic andrequire organic solvents for solubilization and purification.

EXAMPLE 1 Isolation of Human Low Molecular Weight Surfactant ProteinBronchoalveolar Lavage

Bronchoalveolar lavage (BAL) on humans was carried out with a flexiblebronchoscope under local anesthesia. The bronchoscope was wedged in amiddle lobe bronchus and sterile saline solution at 37° C. was instilledin aliquots of 50 ml. The total volume instilled varied between 200 and300 ml. The fluid was gently suctioned back after each instillation andcollected in a siliconized bottle kept on ice. Immediately aftercompletion of the lavate the bottle was transported to the laboratory.

The recovered BAL fluid was strained through a double layer of Dacronnets and the volume was measured. It was centrifuged at 400 g at 4° C.for 5 min and the supernatant was stored at -20° C. until furtheranalyzed.

Amniotic Fluid

Human amniotic fluid, obtained from full term pregnancies at Caesariansections and vaginal deliveries, was filtered through a net and thevolume was measured and the material was stored at -20° C. until furtheranalyzed.

Isolation of hydrophobic proteins from bronchoalveolar and amnioticfluids.

To 300 ml of amniotic or BAL fluids 400 ml of methanol was added and thesolution was mixed by shaking and ultrasonication. 800 ml of chloroformwas added and the mixture was shaken. After filtration the lower phasewas evaporated to dryness and the phospholipid fraction which alsocontains the hydrophobic proteins was isolated by reverse phasechromatograph on Lipidex-5000 in a system of ethylene chloride/methanol1:4 (v/v).

EXAMPLE 2 Analysis of Surfactant Lipoproteins

For determination of amino acid compositions, the proteins were reducedwith dithioerythritol (about 30 nmol/nmol peptide; 37° C.; 2 h) andcarboxymethylated by addition of neutralized iodo (¹⁴ C) acetate (120nmol/nmol peptide; 37° C.; 2 h) in 8M urea, 0.4M Tris/HCX1, 2mM EDTA.Excess reagents were removed by exclusion chromatography on SephadexLH-60 (40×1.1 cm) in chloroform/methanol 1:1 (v/v) containing 5% 0.1MHCl. For analysis of amino acid compositions, samples were hydrolyzed inevacuated tubes for 24 h at 110° C. and for 72 and 120 hrs,respectively, at 150° C., with 6M HCl containing 0.5% phenol. Totalamino acid composition of human surfactant lipoprotein is illustrated inTable 1. Liberated amino acids were analyzed with a Beckman 120Minstrument.

The apparent molecular weight was determined by SDS/polyacrylamide gelelectrophoresis (using a 10% gel containing 8M urea) (22). Molecularweight markers were purchased from BDH Chemicals Ltd (England) andconsisted of horse-heart myoglobin, cleaved by cyanogen bromide.Phosphorus was analyzed according to Bartlett (23).

Preparations for sequence analysis were applied in chloroform/methanol1:2.

Structural Analysis

The lipoprotein fractions were reduced by treatment with dithiothreitol(30 nmol/nmol polypeptide) at 37° C. for 2 h, under nitrogen. Thereduced samples were then ¹⁴ C-carboxymethylated by treatment withneutralized iodo (¹⁴ C)-acetic acid (120 nmol/nmol polypeptide; 37° C.;2 h) and purified by exclusion chromatography on Sephadex LH-60.

Samples for sequence analysis of the ¹⁴ C-carboxymethylated protein wereremoved after solubilization in chloroform/methanol. Samples forcleavages with pepsin were dissolved in 100% formic acid, diluted to 5%formic acid, and then submitted to the enzyme (1:30, enzyme to substrateratio; 37° C.; 2 h). The peptic peptides were separated byhigh-performance liquid chromatography on an Ultropac C-18 column in0.1% trifluoroacetic acid with a linear gradient of acetonitrile.Samples for treatment with CNBr were dissolved in 100% formic acid,diluted to 70%, and then treated with CNBr (0.1 g/ml) at roomtemperature for 24 h. CNBr fragments were separated on Sephadex LH-60 inchloroform/methanol, 1:1 (v/v) containing 5% 0.1M HCl (24).

Gas-phase sequencer analysis was performed by degradation in an AppliedBiosystems 470 A instrument and phenylthiohydantoin detection byreverse-phase high performance liquid chromatography using a HewlettPackard 1090 instrument (25). Samples for liquid-phase sequenceranalysis in a Beckman 890C instrument were applied to glycine-precycledPolybrene (26), and analyzed by a similar high performance liquidchromatography system. Total compositions were obtained by hydrolysiswith 6M HCl/0.5% phenol at 100° C. for 24 h in vacuum. Hydrazinolysiswas performed with anhydrous hydrazine in 100° C. for 6 h in evacuatedtubes (27). Amino acids were quantitated with a ninhydrin-based Beckman121M amino acid analyzer, or with a phenylthiocarbamyl-based highperformance liquid chromatography system (28). N-terminal truncation ofthe surfactant proteins is illustrated in Table 2.

The molecular weight of the surfactant lipoprotein was determined by"Time of flight" mass spectroscopy (Bioion, Uppsala, Sweden) using anacceleration voltage of 18,000 volts. Bovine insulin was used as aninternal reference standard.

The lipoprotein was reduced with dithioerythritol (about 30 mol/molpeptide; 37° C.; 2 h) and analyzed by mass spectrometry. The nonreducedand reduced material had similar molecular weights, about 480 mass unitsgreater than what would be anticipated from the weight of the 35 aminoacids (FIG. 1). To investigate the hypothesis that native surfactantlipoprotein consists of fatty acids covalently linked to the peptidesequence to yield a proteolipid, the nonreduced lipoprotein wasdissolved in chloroform/methanol 1:2 (v/v) and aq. 2M Trimethylamine wasadded to a final concentration of 200 mM.

The mixture was incubated for 4 h at 37° C. Fatty acids were releasedand separated from the polypeptide by exclusion chromatography onSephadex LH-60 in chloroform/methanol 1:1 (v/v) containing 5% 0.1M HCl.Fatty acids, mainly palmitic acid, were recovered.

When the peptide fraction obtained was analyzed by "Time of flight" massspectrometry (FIG. 2) it was evident that the molecular weight haddecreased by about 478 mass units upon trimethylamine treatment.

This reduction in molecular weight corresponds to the loss of twopalmitic acid residues probably esterified to the sulfhydryl groups ofCys 5 and Cys 6.

To confirm that the trimethylamine treatment that released to palmiticacid residues from the surfactant peptide also generated free thiolgroups, the following experiment was made:

Separate samples of the native surfactant lipoprotein were treated withtrimethylamine and any liberated thiol groups were thencarboxymethylated by treatment with dithioerythritol, to preventreoxidation, followed by iodoacetate.

Mass spectroscopical analysis (FIG. 3) demonstrates a gain of about 120mass units compared to the trimethylamine treated peptide fraction. Anincrease in molecular weight of 120 agrees well with carboxymethylationof the thiol groups of Cys 5 and Cys 6.

As a further demonstration of the presence of two esterified palmiticacid residues the native lipoprotein was treated with 0.01M KOH inmethanol/water 98:2 (v/v) at +37° C. for 30 min. The peptide wasrecovered from the unpolar phase (29).

Analysis of this peptide material by "Time of flight" mass spectroscopy(FIG. 4) again shows a reduction in molecular weight of about 480 massunits compared to the native sufactant peptide, demonstrating the lossof two covalently conjugated palmitic acid residues after alkalinehydrolysis.

In FIG. 1 two smaller peaks can be distinguished. One at position -240relative to the main peak probably represents a small proportion of thepeptide material esterified with palmitic acid at only one of the twocysteine residues.

Taken together these data clearly indicate that at least one butpreferentially two palmitic acid residues are covalently conjugated tothe peptide by esterification to the thiol groups of Cys 5 and Cys 6.

EXAMPLE 3 Recombination of Isolated Lipoprotein Fractions with SyntheticPhospholipids

    ______________________________________                                        1.2-Dipalmitoyl-sn-glycero-3-phosphocholine                                                               (DPPC),                                           1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine                                                          (POPC)                                            and                                                                           1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol                                                              (DPPG)                                            ______________________________________                                    

were purchased from Sigma Chemical Co. (St. Louis, Mo.), and were usedwithout further purification. The phospholipids were dissolved inchloroform/methanol 2:1 (v/v) mixed in the proportions DPPC:POPC:DPPG55:35:10 (w/w/w) and used as the surfactant preparation "phospholipids".

To this mixture surfactant lipoprotein dissolved in chloroform/methanol1:2 (v/v) was added, giving a lipoprotein to phospholipid ratio of 1:50.The solvents were evaporated to dryness and the different surfactantpreparations (phospholipids, phospholipids+lipoprotein fraction) weresuspended in saline giving a phospholipid concentration of 10 or 80mg/ml.

EXAMPLE 4 Determination of In-vitro Surface Properties

The surface properties of the lipoprotein-based artificial surfactantwere analyzed with a pulsating bubble instrument (SurfactometerInternational, Toronto, Canada) (30). The surfactant preparations weresuspended at a phospholipid concentration of 10 mg/ml, and the pressuregradient across the bubble wall was recorded at 37° C. during 50% cyclicsurface compression at the rate of 40/min. Surface tension was assessedat maximal and minimal bubble sizes during the 5th cycle and after 1 and5 min of pulsation.

The results of these experiments are shown in FIGS. 5 and 6. Thetracings therein represent pressure gradients across the bubble wall;max and min indicate maximal (radius 0.55 mm) and minimal (radius 0.40mm) bubble size during pulsation at a rate of 40/min. A pressuregradient close to zero at minimal bubble size corresponds to nearly zerosurface tension.

EXAMPLE 5 Effect of Removal of the Fatty Acid Residues on In VitroSurfactant Properties

Native or trimethylamine-treated lipoprotein was mixed with protein-freephospholipids, obtained from lung surfactant by chromatography onSephadex LH-60. These artificial surfactant preparations, containing0-4% of lipoprotein or the protein fraction obtained by the treatmentwere suspended in saline at a phospholipid concentration of 10 mg/ml andanalyzed with a pulsating bubble instrument. The pressure gradientacross the bubble wall was recorded at 37° C. during 50% cyclic surfacecompression at the rate of 40/min. Surface tension at maximal andminimal bubble sizes was determined during the 5th, 40th and 200th cycleof pulsation. The surface adsorption rate was then determined byarresting the pulsation at maximal bubble size and recording the timeinterval until static surface tension had dropped to the level of 30mN/m.

The surfactant preparation containing 2% of the native lipoprotein had arapid adsorption (<2 s) and a minimum surface tension near 0 mN/m. Thecorresponding mixture with trimethylamine treated lipoprotein had slowadsorption (>120 s) and a high minimum surface tension (about 20 mN/m).Thus, acyl residues covalently bound to the polypeptide are essentialfor the physiological activity of the surfactant.

EXAMPLE 6 Preparation of Synthetic Peptides ##STR3## were synthesizedaccording to the stepwise solid phase technique in an Applied BiosystemsModel 430A peptide synthesizer. A phenylacetamidomethyl (PAM) resin wasused as the solid support and the following tert-butyloxycarbonyl(t-Boc) amino acid derivatives were employed: L-Arg-Tosyl,LCys-4-Methyl-Benzyl, L-Lys-Cl-Benzyloxycarbonyl, L-Asn, L-Pro, L-Ala,L-Val, L-Met, L-Ile, L-Leu and Gly. A standard program includingpre-formation of symmetric anhydrides was used for the synthesis. Theresulting peptides were cleaved from the resin and deprotected by thehydrogen fluoride (HF) method and subsequently purified by reverse phasehigh performance liquid chromatography (HPLC). The identity and purityof the final product was assessed by amino acid hydrolysis. (31) Kent,S.B.H. (1980) Ann. Rev. Biochem. 57, 957-989. EXAMPLE 7 Preparation ofThioester Lipopeptide

The conjugation of redestillated Palmitoyl-chloride (Fluka) and thehexapeptide H₂ N-Ile-Pro-Cys-Cys-Pro-Val-COOH was performed as follows:

3 mg (4.8 μmol) of the hexapeptide was dissolved in 500 μl ofchloroform/methanol 1:2 (v/v).

25 μl of 0.5M DTE (12.5 μmol) was added to the solution. The mixture wasflushed with N₂ and incubated 160 minutes in 37° C.

20 μl of 3.3M Palmitoyl-chloride (66 μmol) was added, the mixtureflushed with N₂ and incubated 250 minutes in 37° C.

The acylated peptide was isolated by TLC and its MW was confirmed.

When tested in accordance with the procedure described in Example 4 thelipopeptide performs similarly as the native lipoprotein.

EXAMPLE 8 Determination of In-vitro Surface Properties of ArtificialSurfactants Consisting of Synthetic Phospholipids and Synthesized SP-CPolypeptide (Without Thioester Bound Palmitic Acid)

The SP-C polypeptide was synthesized according to the stepwise solidphase technique in an Applied Biosystems Model 430A peptide synthesizer.A phenylacetamidomethyl resin was used as the solid support and thepolypeptide was cleaved from the resin and deprotected by the hydrogenfluoride method. The polypeptide was extracted from the resin withchloroform/methanol 1:1 (v/v) with and without 5% 0.1M HCl present,yielding about 30% of the sample. The material was dissolved in a smallamount of concentrated formic acid, diluted with chloroform/methanol 1:1(v/v) and purified by Sephadex LH-60 chromatography inchloroform/methanol 1:1 (v/v) containing 5% formic acid. The identityand purity of the final product were assessed by amino acid hydrolysis,sequencer degradation and time-of-flight mass spectrometry.

Various amounts of the purified synthesized polypeptide SP-C wererecombined with the synthetic phospholipid mixture used in Example 3.The phospholipids were dissolved in chloroform/methanol 2:1 (v/v), mixedin the proportions DPPC:POPC:DPPG 55:35:10 (w/w/w) and used as thesurfactant preparation "phospholipids".

Various amounts of synthetic SP-C polypeptide, dissolved in formic acid,was added to different tubes and the acid was evaporated to dryness.After addition of the phospholipids, the organic solvents wereevaporated to dryness and the different surfactant preparations weresuspended in saline at a phospholipid concentration of 10 mg/ml. Theseartificial surfactant preparations, containing 0-20% of synthetic SP-Cpolypeptide, were analyzed with a pulsating bubble instrument. Thepressure gradient across the bubble wall was recorded at 37° C. during50% cyclic surface compression at the rate of 40/min. Surface tension atmaximal and minimal bubble size was determined during the 5th, 40th and200th cycle of pulsation. The surface adsorption rate was thendetermined by arresting the pulsation at maximal bubble size andrecording the time interval until static surface tension had dropped tothe level of 30 mN/m. All preparations had very slow adsorption (>120 s)and high minimum (>20 mN/m) and maximum (>44 mN/m) surface tension(Table 3). The results show that the SP-C polypeptide (without palmitoylresidues) has no effect on surface activity. The slow adsorption andhigh surface tension values preclude the use of these preparations fortreatment of infants.

EXAMPLE 9 Determination of In-vitro Surface Properties of ArtificialSurfactants Consisting of Synthetic Phospholipids and Native orTrimethylamine-treated (=Deacylated) SP-C

The molecular weight of native and trimethylamine-treated SP-C wasdetermined by time-of-flight mass spectrometry. The mass spectrum showedthat native SP-C contained mainly two palmitoyl residues and smallamounts of molecules with one palmitoyl residue. Thetrimethylaminetreated SP-C was purified on Sephadex LH-60 inchloroform/methanol 1:1 (v/v), containing 5% 0.1M HCl and analyzed bytime-of-flight mass spectrometry. This trimethylamine-treated SP-C wascompletely deacylated. Various amounts of native or deacylated SP-C wasrecombined with the synthetic phospholipid mixture used in Example 3.The phospholipids were dissolved in chloroform/methanol 2:1 (v/v), mixedin the proportions DPPC:POPC:DPPG 55:35:10 (w/w/w) and used as thesurfactant preparation "phospholipids".

To these phospholipid mixture various amounts of native or deacylatedSP-C, dissolved in chloroform/methanol 1:2 (v/v), were added. Theorganic solvents were evaporated to dryness and the different surfactantpreparations were suspended in saline at a phospholipid concentration of10 mg/ml. These artificial surfactant preparations, containing 0-4% ofnative or deacylated SP-C, were analyzed with a pulsating bubbleinstrument. The pressure gradient across the bubble wall was recorded at37° C. during 50% cyclic surface compression at the rate of 40/min.Surface tension at maximal and minimal bubble size was determined duringthe 5th, 40th and 200th cycle of pulsation. The surface adsorption ratewas then determined by arresting the pulsation at maximal bubble sizeand recording the time interval until static surface tension had droppedto the level of 30 mN/m. The results show that preparations containingnative SP-C increased the in-vitro surface activity. Thus, aconcentration of 2% of native SP-C in the preparation gave a ratherrapid adsorption (16 s), a minimum surface tension near 0 mN/m and amaximum surface tension about 30 mN/m (Table 4). These results aresimilar to the in-vitro surface activity of natural porcine surfactantpreparations (adsorption <1 s, minimum and maximum surface tensionduring the 200th cycle <3 mN/m and 30 mN/m, respectively) successfullyused for treatment of infants. The adsorption time is somewhat longerthan that obtained with natural surfactant due to the more complex andunsaturated phospholipid mixture in the natural surfactant. Addition ofdeacylated SP-C to the phospholipid mixture did not improve the surfaceproperties. All preparations had very slow adsorption (>120 s) and highminimum (≧18 mN/m) and maximum (≧50 mN/m) surface tension (Table 5). Theresults indicate that the surfactant preparations containing deacylatedSP-C would be ineffective for treatment of infants.

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                  TABLE 1                                                         ______________________________________                                        Human surfactant lipoprotein                                                  110° C.         150° C.                                         24 h         72 h     168 h    24 h   72 h                                    ______________________________________                                        Cys (Cm)                                                                              0.7 (2)                                                               Pro     2.5 (3)                                                               Gly     3.0 (2)                                                               Ala     1.4 (1)                                                               Val     2.4 (10) 2.5 (10) 2.8 (10)                                                                             8.0 (10)                                                                             8.6 (10)                              Met     1.0 (1)                                                               Ile     1.2 (3)  1.3 (3)  1.4 (2)                                                                              2.2 (3)                                                                              1.4 (3)                               Leu     5.1 (7)  5.1 (7)  4.7 (7)                                                                              6.2 (7)                                                                              7.8 (7)                               Phe     0.8 (1)                                                               Lys     1.2 (1)                                                               His     0.8 (1)                                                               Arg     1.2 (2)                                                               Trp     n.d. (1)                                                              ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Hum lipoprotein                                                                             Phe-Gly/Arg-Ile-Pro-                                                                          32%     55%                                                   Gly/Arg-Ile-Pro-                                                                              36%     19%                                                   Ile-Pro-        32%     26%                                                   A               B                                               ______________________________________                                         A: Amniotic fluid                                                             B: Bronchoalvaclar lavage                                                

                  TABLE 3                                                         ______________________________________                                        Surface properties (median values) of synthetic phospholipids                 (10 mg/ml) recombined with different amounts of synthetic                     SP-C polypeptide.                                                             The recordings were obtained with a pulsating bubble at 37° C.,        50% surface compression and rate 40/min. Five experiments                     were performed for each preparation.                                          SP-C                                                                          POLY-    ADSORP-   SURFACE TENSION (mN/m)                                     PEPTIDE  TION      5th cycle 40th cycle                                                                            200th cycle                              (%)      (s)       .sup.τ min                                                                       .sup.τ max                                                                     .sup.τ min                                                                     .sup.τ max                                                                     .sup.τ min                                                                     .sup.τ max                  ______________________________________                                         0       >120      20     52   20   55   22   58                               1.0     >120      32     65   29   63   22   58                               2.0     >120      27     61   24   58   20   56                               4.0     >120      27     51   24   50   21   49                              10       >120      30     51   29   50   26   50                              20       >120      36     47   35   45   34   44                              ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Surface properties (median values) of synthetic phospholipids                 (10 mg/ml) recombined with different amounts of native SP-C.                  The recordings were obtained with a pulsating bubble at 37° C.,        50% surface compression and rate 40/min. Five experiments                     were performed for each preparation.                                          NATIVE   ADSORP-   SURFACE TENSION (mN/m)                                     SP-C     TION      5th cycle 40th cycle                                                                            200th cycle                              (%)      (s)       .sup.τ min                                                                       .sup.τ max                                                                     .sup.τ min                                                                     .sup.τ max                                                                     .sup.τ min                                                                     .sup.τ max                  ______________________________________                                        0        >120      22     60   22   58   21   54                              1.0        88      12     41   12   38   11   52                              2.0        16      13     33   11   30    1   32                              4.0        26      12     41    6   39    1   35                              ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Surface properties (median values) of synthetic phospholipids                 (10 mg/ml) recombined with different amounts of deacylated                    SP-C.                                                                         The recordings were obtained with a pulsating bubble at 37° C.,        50% surface compression and rate 40/min. Five experiments                     were performed for each preparation.                                          DEACY-                                                                        LATED    ADSORP-   SURFACE TENSION (mN/m)                                     SP-C    TION       5th cycle 40th cycle                                                                            200th cycle                              (%)     (s)        .sup.τ min                                                                       .sup.τ max                                                                     .sup.τ min                                                                     .sup.τ max                                                                     .sup.τ min                                                                     .sup.τ max                  ______________________________________                                        0       >120       25     56   27   54   23   53                              1.0     >120       19     55   22   56   18   56                              2.0     >120       21     54   22   54   21   54                              4.0     >120       39     52   37   50   27   50                              ______________________________________                                    

We claim:
 1. A purified lipoprotein possessing pulmonary surfactantactivity comprising an alveolar polypeptide or protein including theamino acid sequence:Phe-Gly-Ile-Pro-Cys-Cys-Pro-Val-His-Leu-Lys-Argand,covalently bound thereto, one or two fatty acid residue(s).
 2. Thelipoprotein according to claim 1 wherein said fatty acid residue isderived from a fatty acid having from 14-22 carbon atoms.
 3. Thelipoprotein according to claim 2 wherein said acid is palmitic, stearic,oleic, linoleic or linolenic acid.
 4. The lipoprotein according to claim3 wherein said acid is saturated.
 5. The lipoprotein according to claim4 wherein said acid is palmitic or stearic acid.
 6. The lipoproteinaccording to claim 4 wherein said fatty acid is palmitic acid.
 7. Thelipoprotein according to claim 1 comprising two fatty acid residues.